The objective of Project 1 is to characterize the important role of repressive histone methylation in nucleus accumbens (NAc) and prefrontal cortex (PFC) in mediating depression- and antidepressant-like responses in animal models. We have demonstrated downregulation of di-methylation of Lys 9 of histone H3 (H3K9me2) in NAc and PFC after several forms of chronic stress, which is mediated by repression of G9a and GLP, the histone methyltransferases which catalyze this epigenetic state. We have also demonstrated that such adaptations mediate pro-depression-like effects, while augmenting H3K9me2 exerts potent antidepressant actions. We will now extend our considerable preliminary findings in several ways. We will utilize ChlP-Seq to map H3K9me2 binding genome-wide. While global levels of H3K9me2 are reduced in NAc by chronic stress, many genes show induction of G9a/H3K9me2 binding, indicating the existence of complex mechanisms that control this repressive histone methylation mark at particular genes. Our hypothesis is that the chromatin state at individual genes helps determine which genes show reduced vs. increased H3K9me2 binding overlaid on global decreases in the enzymatic machinery involved. In conjunction with the Chromatin and Gene Analysis Core, we will also gain crucial information concerning the relationship between H3K9me2 and several other chromatin modifications in the brain in vivo, and why certain genes show stable chromatin changes over the life cycle of the animal. By exploring the involvement of the transcription factor CREB, which we have found interacts with G9a/H3K9me2 as part of a pathological molecular circuit in NAc to drive susceptibility to stress, we expect to gain unprecedented insight into mechanisms of gene regulation in depression models. We will then focus on a small number of target genes for H3K9me2 and characterize their involvement in depression-related phenomena. Many of our key findings observed to date in animal models have already been validated in the NAc and PFC of depressed humans, as discussed in Project 4, emphasizing the translational nature of our Center. These studies provide a template for the analysis of the roles played by other key histone modifications in depression. RELEVANCE (See instructions): Depression has a lifetime risk of ~15% for the U.S. general population, yet available antidepressant therapies are based on serendipitous discoveries over 6 decades ago, and fully treat <50% of all affected individuals. An improved understanding of the molecular basis of depression will lead to improved treatments and diagnostic tests-a high priority for the National Institutes of Health.